Project Summary Project 4 will examine the pathogenesis of ETEC diarrhea using the human enteroid monolayer model. Acute ETEC diarrhea is one of the four leading causes of diarrhea and diarrheal death world wide. ETEC and its major virulence factors, STa and LT, have been extensively studied in animal intestine, human colon cancer cells lines, but there is minimal mechanistic information from human intestine. Moreover, additional virulence factors have been identified, including the bacterial protease and mucinase EatA, the role of which in ETEC diarrhea has only been partially defined. Since ST-containing ETEC were highly associated with clinical disease, we will particularly focus on the role of STa and EatA in ETEC pathogenesis. A human proximal small intestinal enteroid model is being developed using ETEC producing its virulence factors STa, LT and EatA., with the model demonstrating microcolonies of ETEC attached to human enteroid BB, production of STa in the luminal fluid and activation by STa of GCC/cGMP. Aim 1 will characterize the roles of STa in pathogenesis using isogenic mutants and purified Sta, including mutants being proposed as toxoids for vaccine development. Effects will be defined on Na absorptive and Cl secretory transport proteins that contribute to other diarrheas, changes in tight junctions and other BB and BLM structural proteins, as well as epithelial cytokine release. Biochemical and proteomics approaches will be used to further characterize the signaling complexes affected by STa binding to BB GCC, which are involved in NHE3 inhibition, thus identifying multiple additional potential drug targets to treat acute ETEC diarrhea. Aim 2 will examine the role of EatA in ETEC pathogenesis determining its effects on release of other ETEC virulence factors, whether it alters intestinal transport processes to contribute directly to diarrhea, and if it cleaves intestinal mucins including the consequences of cleaving these mucins. Aim 3 will determine if ETEC produces longer lasting effects on the enteroids that could contribute to environmental enteropathy By developing an acute ETEC diarrhea model in normal human enteroids, insights in pathobiology of human ETEC diarrhea and identification of new targets to develop drug treatment will be identified, which will complement approaches by others to develop ETEC vaccines.